PROJECT SUMMARY According to the Centers for Disease Control and Prevention, insufficient sleep is an epidemic that poses significant clinical and economic impacts. Poor sleep can be caused by impaired sleep homeostasis which regulates sleep need as a function of prior wakefulness. Therefore, determining the cellular basis of sleep homeostasis is necessary to understand underlying causes of abnormal sleep. The biological substrates of sleep homeostasis are incompletely understood, but glial astrocytes may play a central role. Astrocytes are found throughout the brain and modulate compensatory responses to sleep loss. Because astroglial chemical signaling is mediated by changes in intracellular calcium, I hypothesize that astroglial intracellular calcium dynamics contribute to the accumulation and discharge of sleep need. I will test this hypothesis in two Aims: 1) Characterize astroglial calcium dynamics in spontaneous sleep and in response to sleep deprivation; 2) Determine the extent to which astroglial calcium signaling contributes to sleep homeostasis. Intracellular calcium activity in astroglial somata and processes will be measured in vivo with a head-mounted epifluorescent microscope and two-photon microscopy in unanesthetized mice expressing the genetically encoded calcium indicator GCaMP6f selectively in astrocytes. Astroglial calcium dynamics will be simultaneously recorded with sleep-wake behavior, as determined by electroencephalography and electromyography, under physiological conditions, in response to sleep deprivation, and following genetic depletion of astroglial intracellular calcium stores. These studies will be the first to describe astroglial calcium activity in conjunction with electroencephalographic determination of arousal state. The results of these experiments will provide new, mechanistic insights into astroglial mediation of sleep homeostasis.